Sepsis is a life-threatening medical condition that can be brought on by infection or trauma. The symptoms of sepsis can include chills, profuse sweating, fever, weakness, or hypotension, followed by leukopenia, intravascular coagulation, shock, adult respiratory distress syndrome, multiple organ failure, and often, death. R. Ulevitch, et al., J. Trauma 30: S189-92 (1990).
The lipopolysaccharides ("LPS"; also, "endotoxins") that are typically present on the outer membrane of all gram-negative bacteria are among the most studied and best understood sepsis-inducing substances. While the precise chemical structures of LPS molecules obtained from different bacteria may vary in a species-specific fashion, a region called the lipid A region is common to all LPS molecules. E. Rietschel et al., in Handbook of Endotoxins, 1: 187-214, eds. R. Proctor and E. Rietschel, Elsevier, Amsterdam (1984). This lipid A region is responsible for many, if not all, of the LPS-dependent pathophysiologic changes that characterize sepsis.
LPS is believed to be a primary cause of death in humans afflicted with gram-negative sepsis. van Deventer et al., Lancet, 1: 605 (1988); Ziegler et al., J. Infect. Dis., 136: 19-28 (1987). Treatment of patients suffering from sepsis and gram-negative bacteraemia with a monoclonal antibody against LPS decreased their mortality rate. Ziegler et al., N. Eng. J. Med., 324: 429 (1991).
Sepsis is also caused by gram-positive bacteria. Bone, R. C. Arch. Intern, Med., 154: 26-34 (1994). The activation of host cells can originate from gram-positive cell walls or purified cell components such as peptidoglycan and lipoteichoic acid. Such substances induce a similar pattern of inflammatory responses to those induced by LPS. Chin and Kostura, J. Immunol. 151: 5574-5585 (1993); Mattson et al., FEMS Immun. Med. Microbiol. 7: 281-288 (1993); and Rotta, J. Z. Immunol. Forsch. Bd.: 149: 230-244 (1975).
LPS and gram-positive cell wall substances cause polymorphonuclear leukocytes, endothelial cells, and cells of the monocyte/macrophage lineage to rapidly produce and release a variety of cell products, including cytokines, which are capable of initiating, modulating or mediating humoral and cellular immune responses and processes.
One particular cytokine, alpha-cachectin or tumor necrosis factor (TNF-.alpha.), is apparently a primary mediator of septic shock. Beutler et al., N. Eng. J. Med., 316: 379 (1987). Intravenous injection of LPS into experimental animals and man produces a rapid, transient release of TNF-.alpha.. Beutler et al., J. Immunol., 135: 3972 (1985); Mathison et al., J. Clin. Invest. 81: 1925 (1988). Pretreatment of animals with anti-TNF-.alpha. antibodies can modulate septic shock. Beutler et al., Science, 229: 869, (1985); Mathison et al., J. Clin. Invest. 81: 1925 (1988).
Molecular receptors that can combine with sepsis-inducing substances, and that once combined, initiate certain chemical reactions, play a critical role in the etiology of the symptoms of sepsis. CD14 is a 55-kD glycoprotein expressed strongly on the surface of monocytes and macrophages, and weakly on the surface of granulocytes, such as neutrophils. S. M. Goyert et al., J. Immunol. 137: 3909 (1986). A. Haziot et al., J. Immunol. 141: 547-552 (1988); S. M. Goyert et al., Science 239: 497 (1988).
The cDNAs and the genes for human and murine CD14 have been cloned and sequenced. E. Ferrero and S. M. Goyert, Nuc. Acids Res. 16: 4173 (1988); S. M. Goyert et al., Science 239: 497 (1988); M. Setoguchi et al., Biochem. Biophys. Acta 1008: 213-22 (1989). CD14 is linked by a cleavable glycosyl phosphatidyl inositol tail A. Haziot et al., J. Immunol. 141: 547-552 (1988)! to the exoplasmic surface of mature monocytes, macrophages, granulocytes and dendritic reticulum cells, or renal nonglomerular endothelium, and of hepatocytes in rejected livers.
CD14 mediates responses by binding to LPS. Complexes of LPS and sCD14 exhibit a 1:1 stoichiometry (Hailman, E., et al., J. Exp. Med. 179: 269-277 (1994)), and these complexes initiate TNF-.alpha. production in monocytes (Dentener, M. A., et al., J. Immunol. 7: 2885-2891 (1993)), IL-6 production in astrocytes (Frey, E., et al., J. Exp. Med. 176: 1665-1671 (1992)), production of adhesion molecules in endothelial cells (Frey, E., et al., J. Exp. Med. 176: 1665-1671 (1992)) and activation of leukocyte integrins in PMN (Hailman, E., et al., J. Exp. Med. 179: 269-277 (1994)). Spontaneous binding of LPS to CD14 is slow, but this binding may be dramatically accelerated by LBP. LBP acts in a catalytic fashion, with one molecule of LBP transferring hundreds of LPS molecules to hundreds of CD14 molecules.
Other experiments have shown that cell activation can also be induced by interaction of CD14 with components of gram-positive bacteria such as B. subtilis, S. aureus, and S. mitus (Pugin et al., Immunity 1: 509-516 (1994). Furthermore, interaction of CD14 with lipoarabinomannan from the cell wall of Mycobacterium tuberculosis also induces cellular activation in a CD14-dependent fashion (Zhang et al., J. Clin. Invest. 91: 2076-2083 (1993); Pugin et al., Immunity 1: 509-516 (1994)). These studies suggest that CD14 is a receptor which recognizes a wide variety of bacterial structures. Interaction of CD14 with these structures initiates host inflammatory responses.
Several neutralizing monoclonal antibodies (mAbs) to CD14 have been shown to antagonize cellular responses to LPS in vitro (Wright, S. D., et al. Science 249: 1431-1433 (1990); Hailman, E., et al. J. Exp. Med. 179, 269-277 (1994); Frey, E. A., et al. J. Exp. Med. 176, 1665-1671 (1992); Arditi, M., et al. Infect. Immun. 61, 3149-3156 (1993); Wright, S. D., et al. J. Exp. Med. 173, 1281-1286 (1991); Dentener, M. A., et al. J. Immunol 150, 2885-2891 (1993); Grunwald, U., et al. J. Immunol Methods 155, 225-232 (1992)) and in vivo (Leturcq, D. J., et al. Satellite Meeting of the 3rd Conference of the International Endotoxin Society 22 (Abstract) (1994)); and Wright, et al., Science 90: 1431-1433 (1990). Additional in vivo data have demonstrated that animals injected with CD14 neutralizing monoclonal antibodies become hyporesponsive to LPS and mice lacking CD14 fail to respond to LPS. These experiments suggest that the release of inflammatory cytokines can be blocked by preventing the interaction of LPS with membrane CD14.
CD14 has also been shown to exist as a soluble protein found in normal sera or urine of nephrotic patients. Recent evidence has shown that sCD14 enables LPS-dependent responses in cells which lack membrane CD14, i.e., endothelial cells and epithelial cells. In these cells types, sCD14 in conjunction with LPS promotes inflammatory cytokine release and upregulation of adhesion molecules.
Interestingly, high concentrations of sCD14 have been shown to block inflammatory cytokine release from monocytes in a whole blood assay. Presumably, the beneficial effect of sCD14 in this assay arises from its ability to divert LPS away from mCD14 on macrophages and PMNs. Thus sCD14, like CD14 neutralizing monoclonal antibodies, could be useful in preventing LPS interactions in mCD14. However, the utility of sCD14 to treat LPS-mediated inflammatory disorders is limited by its other property of eliciting inflammatory cytokines in endothelial cells. Thus, a sCD14 molecule which retained its ability to bind LPS, yet did not activate endothelial cells should have superior properties in treating inflammation.
Monoclonal antibodies may be a useful tool to help identify domains in sCD14 required for cell activation. We have demonstrated that mAbs MEM-18 and 3C10 recognize a sCD14 mutant truncated at amino acid 152, indicating that epitopes for these two mAbs are within the first 152 amino acids (Juan, T. S. -C., et al. J. Biol. Chem. 270, 1382-1387 (1995)). We further localized the epitope of MEM-18 between amino acids 57 and 64 and found that this region is also essential for LPS binding (Juan, T. S. -C., et al. J. Biol. Chem. 270, 5219-5224 (1995)). Deletion of this region not only disrupted binding of MEM-18, but also binding of LPS.
The epitope for mAb 3C10 defines another functional domain of CD14. This mAb appears to recognize a different region from that of MEM-18 (Juan, T. S. -C., et al. J. Biol. Chem. 270, 1382-1387 (1995)). Binding of monoclonal antibody 3C10 to sCD14 does not affect LPS binding to sCD14 (Juan, T. S. -C., et al. J. Biol. Chem. 270, 5219-5224 (1995)), suggesting that this epitope may be involved in a cellular function other than LPS binding.
For the preceding reasons, it is an object of this invention to develop methods and therapies for the effective treatment, including prevention, for symptoms of inflammatory conditions, including sepsis. It is also an object of this invention to develop methods and therapies for the effective protection of individuals who are at risk of becoming afflicted by the symptoms of inflammation, including sepsis.
It is another object of this invention to develop methods and therapies for the effective treatment, including prevention, of symptoms of diseases that are mediated by LPS, gram-negative bacteraemia, gram-positive cell components, gram-positive bacteraemia, mycobacterial lipoarabinomannan, mycobacterial infections and/or CD14. Such diseases include ARDS, septic shock, acute pancreatitis, acute and chronic liver failure, intestinal or liver transplantation, inflammatory bowel disease, graft vs. host disease in bone marrow transplantation and tuberculosis.